Super-sensitive detection of universal markers of allograft rejection Confidential Principal Investigator: Shafer, David A., PhD PROJECT SUMMARY Over 35,000 organ transplants are performed annually in the US, with kidney, liver, heart, and lung transplants being most common. Transplant recipients are normally prescribed immunosuppressants, corticosteroids, or other medications to prevent allograft rejection; however, rejection eventually occurs in up to one third of recipients. Monitoring rejection facilitates timely therapy by switching to a new drug, adding another drug, or adjusting the dose of the current medications. Tissue biopsies are the gold standard for diagnosing allograft rejection, although they are invasive, may result in medical complications, and often yield inadequate specimens. Currently, the AlloMap test (CareDx) is the only FDA-approved molecular diagnostics test for identifying the risk of acute cell rejection in transplant recipients. The AlloMap test does not directly measure donor-derived cell-free DNA (ddcfDNA), has a low positive-predictive value, and does not detect antibody- mediated rejection. Thus, a genuine clinical need exists for accurate methods that enable inexpensive, non- invasive ?liquid biopsy? biomarker testing via plasma samples to quickly identify subjects at risk of rejection. The overall objective of this project is to develop a real-time PCR SNP profiling test for detecting ddcfDNA in the plasma of transplant recipients, as an early indicator of rejection. Our primary system, the internal DNA- Detection Switch (iDDS) probe system, comprises two interacting components: a fluor-labeled probe, and a quencher-labeled antiprobe that is nearly complementary to the probe. This unique probe system shows superior single-base discrimination over a much wider annealing-temperature range (10?30C) than common methods. Recently, we merged our iDDS probe technology with our Wild Terminator (WTx) methods that enable detection of rare mutants (0.1?0.01% frequency) by blocking amplification of the wild-type sequence. The combined method showed greater sensitivity in detecting circulating EGFR variants from lung cancer patients than the FDA-approved cobas EGFR Mutation Test, v2. Our higher sensitivity and specificity will enable development of platform-independent, liquid biopsy assays. Our Specific Aims are: 1) to develop dual-iDDS probe-based SNP profiling assays for donor-derived SNPs, and 2) to develop WTx-iDDS probe assays for quantitating SNPs in ddcfDNA. In Aim 1, we will develop dual-iDDS probe profiling assays for two SNP variants at each of 20 loci, at sites where the global minor allele frequency is nearly 0.5. Selecting SNPs with nearly equal major/minor allele frequencies will greatly simplify the process of detecting a spike in ddcfDNA levels in the blood of transplant recipients before rejection occurs. In Aim 2, we will develop WTx assays that selectively amplify the 20 targeted SNP alleles, and validate key test features (linearity, analytical sensitivity, comparison with NGS, and precision) required for a 510(k) submission. We will require that each WTx-iDDS probe assay can detect the target SNP at a frequency of 0.1%, which is below the threshold observed in patients before allograft rejection. Success in Phase I will justify expanded Phase II assay-validation studies in preparation for filing for FDA approval.